Available online at www.pelagiaresearchlibrary.

com

Pelagia Research Library

Advances in Applied Science Research, 2012, 3 (6):3950-3955

ISSN: 0976-8610
CODEN (USA): AASRFC

Isolation, characterization & biomass production of Trichoderma viride using

various agro products- A biocontrol agent

Mridula Khandelwal, Sakshi Datta, Jitendra Mehta*, Ritu Naruka, Komal Makhijani,
Gajendra Sharma, Rajesh Kumar and Subhas Chandra

Microbiology and Pathology Division, Vital Biotech Research Institute, University of Kota,
Kota- 324009 (Rajasthan) India.
_____________________________________________________________________________________________

ABSTRACT

In the recent years, the environmental contamination caused by excessive use of chemical pesticides increased the
interest in integrated pest management, where chemical pesticides are substituted by bio-pesticides to control plant
pests and plant diseases. Trichoderma spp, is a potential fungal biocontrol agent against a range of plant
pathogens. Present study deals with use of household waste, vegetable waste and other wastes and assessment for
their suitability as substrates for mass multiplication of Trichoderma viride and develops effective production
methodology which can be easily adopted. For the production of biomass of fungal grain media, organic media and
non-synthetic media have been used. The maximum biomass production of Trichoderma spp. was observed in pulses
(19.98 gm in 250 ml).

Key Words: Pathogenic fungi, Grain Medium, Non Synthetic Medium and Organic Medium, Biomass production.
_____________________________________________________________________________________________

INTRODUCTION

The conventional chemical pesticides has not only enhanced the food production, but also adversely affected the
environment and non-target organisms. Due to the side-effects of chemical pesticides, the sustainable crop
production through eco-friendly pest management is essentially required in recent scenario. Among the several
micro-organisms viz., bacteria, fungi, virus, protozoans and entomopathogenic nematodes, a few have been
systematically studied for their effective beneficial characteristics. Trichoderma viride is very promising method
against soil borne plant parasitic fungi. The fungal pathogens play a major role in the development of diseases on
many important field and horticultural crops; resulting in severe plant yield losses. Intensified used of fungicides has
resulted in accumulation of toxic compound potentially hazardous to human and environment an also in the buildup
of resistance of the pathogens. In order to tackle these national and global problems, effective alternatives to
chemical control are being employed. Biological control by an antagonism is a potential, no chemical and
ecofriendly approach for managing plant diseases. [1]. Trichoderma is one of the common fungal biocontrol agent,
is being used world wide for suitable management of various foliar and soil borne plant pathogens.[2] Biocontrol
agents like Trichoderma spp. are acclaimed as effective, ecofriendly and cheap, nullifying the ill effects of
chemicals. Therefore, of late, these biocontrol agents are identified to act against on array of important soil borne
plant pathogens causing serious diseases of crops. Therefore considering the cost of chemical pesticides and
hazardous involves, biological control of plant diseases appears to be an effective and ecofriendly approach being
practice world over. Further biological control strategy is highly compatible with sustainable agriculture and has a
major role to play as a component of integrated pest management (IPM) programme. Large scale production, along

3950
Pelagia Research Library
Jitendra Mehta et al Adv. Appl. Sci. Res., 2012, 3(6):3950-3955
_____________________________________________________________________________
with shelf life and establishment of bioagents in targeted niche, determine the success of biological control.
Therefore cost effective large scale production, shelf life of formulation, establishment of bioagent in to targeted
niche and consistency in disease control are the primary concern with augmentative biological control. Adaptation
of technology in the biocontrol arsenal needs to be investigated. Development of acceptable easily prepared and cost
effective formulations for delivery should be major goal.

Present study was carried out to evaluate grains such as rice, wheat, pulses and maize at different temperatures, and
naturally available solid media such as carrot, ladyfinger, jack seeds, rice husk, and saw dust for the biomass
production of Trichoderma viride .For mass multiplication of bio agent through liquid state fermentation technology
an enormous quantity of spore biomass is needed. Various substrates like sugarcane baggase, fruit juice waste,
vegetable waste, rotten wheat grains etc. are being used for mass multiplication of Trichoderma viride with various
degree of success. [3]Moreover a huge amount of solid waste like sugarcane baggase, fruit juice wastes, vegetable
waste and rotten wheat grains increasing pollution and disposal problems. Therefore looking towards need for large
scale cost effective production of ecofriendly biopesticide, present investigation is carried out to evaluate locally
available cheaper substrates for mass multiplication of Trichoderma viride for sustainable environment and
sustainable agriculture.

MATERIALS AND METHODS

Isolation and Identification of T. viride

Fungal species Trichoderma viride was isolated from soil samples by using potato dextrose agar (PDA) medium.
Samples were inoculated over plates by multiple tube dilution technique (MTDT) and the plates were incubated at
26˚C for 4 days. The fungal colonies which were picked up and purified by streaking and incubated at 26˚C for 7-8
days. Green conidia forming fungal bodies were selected and microscopic observation was identified to be
Trichoderma viride. The culture was maintained on PDA slants [4].

Maintenance of culture
A loopful of inoculum from sub cultured plates of Trichoderma viride were transferred to Potato Dextrose Agar
(PDA) slants and maintained as pure culture.

For laboratory studies, the fungus was cultured on PDA medium. The medium was sterilized at 15 psi for 30 min in
autoclave, poured to sterilized plates, cooled and inoculated with pure culture of the fungus under aseptic conditions.
The plates were then incubated at room temperature (26±2°C) for ten days. After complete sporulation, conidia from
the medium were harvested by washing them thoroughly with sterilized water containing Tween-20 (0.2%) for
immediate use. Otherwise, spores were harvested with the help of a small sterile metal spatula. Harvested conidia
were air dried under laminar air flow and stored in a small air tight screw cap vials (10 cm with 2.5 cm diameter) in
refrigerator at 4°C before using for further studies. Colony forming units (cfu) were estimated by plating technique.
Suspension of spores was made using distilled water with Tween-20 (0.2%) and filtered through a double layered
muslin cloth. Spore count was made using a double rolled Neubauer’s haemocytometer after necessary serial
dilutions under phase contrast microscope. From the stock solution, further dilutions were made to obtain the
required concentrations for further studies.

Grain Medium
Three Grains viz rice, wheat and pulses were used for estimating the biomass of Trichoderma viride at 26 ˚C. 20 g
of each grain was washed well and boiled in distilled water for 1 hr. and then mesh properly and filter it, now
makeup 1 liter with distilled water. Now these grain mediums were packed separately in individual 500 ml conical
flask for Trichoderma viride. They were plugged with cotton wool and autoclaved at 15 psi for 1hr at 121˚C. After
cooling, 1 gm of the fungal culture was inoculated into each flask, separately. All these action were done under
laminar air flow chamber. They were incubated in BOD incubator at 26˚C for 3 weeks. To avoid clumping, after 7
days of inoculation, the flasks were shaken vigorously to separate the culture and to break the mycelial mat.

After 14 days of incubation, the mycelial mat appeared in flasks. Now it was grow well for 21 days. The flasks were
shaken in mechanical shaker for 10 min. The suspension was filtered through double layered muslin cloth and then
taken biomass in each grain medium.

3951
Pelagia Research Library
Jitendra Mehta et al Adv. Appl. Sci. Res., 2012, 3(6):3950-3955
_____________________________________________________________________________
Non Synthetic Medium
Three Non Synthetic Medium viz saw dust, rice brawn and carrot were used for estimating the biomass of
Trichoderma viride at 26 ˚C. 20 g of each grain was washed well and boiled in distilled water for 1 hr and then
mesh properly and filter it, now makeup 1 liter with distilled water. Now this Non Synthetic Medium was packed
separately in individual 500 ml conical flask for Trichoderma viride. They were plugged with cotton wool and
autoclaved at 15 psi for 1hr at 121˚C .After cooling, 1 gm of the fungal culture was inoculated into each flask,
separately. All these procedures were done under laminar air flow chamber. They were incubated in BOD incubator
at 26˚C for 3 weeks. To avoid clumping, after 7 days of inoculation, the flasks were shaken vigorously to separate
the culture and to break the mycelial mat. After 14 days of incubation, the mycelial mat appears again in flasks. Now
it was grow well for 21 days. The flasks were shaken in mechanical shaker for 10 min. The suspension was filtered
through double layered muslin cloth and then taken biomass in each Non Synthetic Medium.

Organic Medium
Three Organic Medium viz yeast, potato dextrose and jaggery were used for estimating the biomass of Trichoderma
viride at 26 ˚C. 20 g of each grain was washed well and boiled in distilled water for 1 hr. and then mesh properly
and filter it, now makeup 1 liter with distilled water. Now this Organic Medium was packed separately in individual
500 ml conical flask for Trichoderma viride. They were plugged with cotton wool and autoclaved at 15 psi for 1hr at
121˚C. After cooling, 1 gm of the fungal culture was inoculated into each flask, separately. All these procedures
were done under laminar air flow chamber. They were incubated in BOD incubator at 26˚C for 3 weeks. To avoid
clumping, after 7 days of inoculation, the flasks were shaken vigorously to separate the culture and to break the
mycelial mat. After 14 days of incubation, the mycelial mat appears again in flasks. Now it was grow well for 21
days. The flasks were shaken in mechanical shaker for 10 min. The suspension was filtered through double layered
muslin cloth and then taken biomass in each Organic Medium.

RESULTS AND DISCUSSION

In the present study, several naturally available substrates of both solid & liquid media were tested for mass
multiplication of Trichoderma viride. The success of biological control depends not only the isolation,
characterization & pathogenicity, but also on the successful mass production of the fungal agent in laboratory. Large
scale availability of the pathogen is a primary requirement in the biocontrol programmed. For a successful integrated
pest management programme, the agents like the pathogenic fungi should be amenable to easy & cheap mass
multiplication. Current research efforts were directed at selecting native bio control fungi, characterizing them
assessing their virulence and developing a formulation for them. The results obtained are discussed herein.

Grain Medium
Among the grain media, pulses medium produced significantly higher 19.98 gm per 250 ml of biomass production
was recorded in Trichoderma viride. Abundance of minerals in the pulses medium may enhance the growth of fungi.
Rice and wheat medium also supported the growth of both the tested fungi.

Non Synthetic Medium

Among the Non Synthetic Medium, Beetroot produced significantly higher 14.32 gm per 250 ml of biomass
production was recorded in Trichoderma viride respectively.

Organic Medium
Among the Organic Medium, Potato produced significantly higher 16.05 gm gm per 250 ml of biomass production
was recorded in Trichoderma viride respectively.

In all three categories other media also supported biomass production in all tested fungi (Table-1, Figure-1 A-H, and
Figure-2).

3952
Pelagia Research Library
Jitendra Mehta et al Adv. Appl. Sci. Res., 2012, 3(6):3950-3955
_____________________________________________________________________________
Figure-1 (A-H) Biomass production of Trichoderma viride fungi

A. Microscopic view of Trichoderma viride. B. Mother culture of Trichoderma viride.

C. Slants of Trichoderma viride. D. 3 weeks in Pulses media

3953
Pelagia Research Library
Jitendra Mehta et al Adv. Appl. Sci. Res., 2012, 3(6):3950-3955
_____________________________________________________________________________
Figure-2 Biomass production of Trichoderma viride fungi observed in various media.

Table-1 Biomass production of Trichoderma viride

Microorganism
Media
(Temperature 26 ˚C, Duration 3 weeks)
Grain Trichoderma viride
Frequency of biomass
Biomass (gm/ 250 ml)
Rice Growth (%)
10.90 62
Wheat 12.29 64
Pulses 19.98 90
Non Synthetic
Saw dust 10.54 63
Rice Brawn 9.34 58
Beet root 14.32 80
Organic
Beef 9.88 60
Potato 16.05 85
Jaggery 13.39 65

CONCLUSION

The excess use of chemical pesticides not only polluted the environment but also deteriorated the overall fertility of
the soil. There can be benefits using pesticides at initial level but later use can counterproductively increase pest
resistance and kill the natural enemies of pests and also adversely affect the fertility of soil. In turn, bio-pesticides
not only increase the fertility of soil, but also are eco-friendly and do not affect the other beneficial microorganisms.
The pathogenic fungi are considered natural mortality agents and environmentally safe bio-pesticides. Bio-pesticides
are more effective than chemical pesticides in long term use, and also cost effective. Pathogenic fungi have been
reported as an excellent bio-pesticide. From all above methods we were concluded that pulses showed a high growth
rate for biomass production of entomopathogenic fungi (Trichoderma viride) as compare to beetroot and potato
medium (Table-1, Figure-1 A-H, and Figure-2)..

Acknowledgement
We are grateful to Department of Microbiology and Pathology division, Vital Biotech Research Institute, Kota for
providing laboratory facilities and also thankful to Mr. Jitendra Mehta of Vital Biotech for sincere efforts in writing

3954
Pelagia Research Library
Jitendra Mehta et al Adv. Appl. Sci. Res., 2012, 3(6):3950-3955
_____________________________________________________________________________
this research paper. We are also grateful to Mrs Mridula Khandelwal, Miss Sakshi Datta, Miss Ritu Naruka, Miss
Komal Makhijani, Mr. Gajendra Sharma, Mr. Rajesh Kumar and Mr. Subhas Chandra for their continuous team
work. We would also like to thank Mrs. Jaya Mehta and staff members of Vital Biotech Research Institute for
encouragement and we would like to thank the reviewers of this paper for their excellent comments.

REFERENCES

[1] Bailey, D.J., A. Kleczkowski and C.A. Gilligan, New Phytol., 2004,161: 569-576.
[2] Dominguesa F.C., J.A. Queiroza, J.M.S. Cabralb and L.P. Fonsecab, Enz. Microbial Technol., 2000, 26: 394-
401.
[3] Esposito, E. and M. da Silva, Crit. Rev. Microbiol., 1998, 24: 89-98.
[4] Gamal, M. Abdel-Fattah, Yasser M. Shabana, Adel E. T. harzianum: Mycopathology, 2007, 164: 81-89.
[5] Bissett, J. Can. J. Bot. 1991, 69:2418-2420.
[6] Bissett, J., Can. J. Bot,. 1992, 70:639-641.
[7] Bissett, J., Szakacs, G., Nolan, C. A., Druzhinina, I., Gradinger, C., and Kubicek, C. P. Can. J. Bot., 2003,
81:570-586.
[8] B.K. Mishra, Archives of Applied Science Research, 3 (2):361-369.
[9] Brian, P. W., Curtis, P. J.,., Ann. Appl. Biol. 1946, 33:190-200.
[10] Brian, P. W., and Hemming, H. G., Ann. Appl. Biol. 1945, 32: 214-220.
[11] Brian, P. W., and McGowan, J. C. Viridin: Nature (London) .1945, 156:144.
[12] Mehta J, Sain M, Mathuriya BL, Naruka R, Kavia A, Sharma DR, Asian Journal of Plant Science and
Research, 2012, 2 (3): 364-368.
[13] Mehta J, Naruka R, Sain M, Dwivedi A, Sharma D, Mirza J, Asian Journal of Plant Science and Research,
2012, 2 (4):518-523.
[14] Mehta J, Kaushal N, Sen P, Sharma D, Dhillon MD, Mathuriya BL, European Journal of Experimental Biology,
2012, 2 (4):1278-1283.
[15] Tanwer B S, Advances in Applied Science Research, 2011, 2 (1): 208-213.
[16] Bhosale U P, Asian Journal of Plant Science and Research, 2011, 1 (3):23-27.

3955
Pelagia Research Library